Process for producing copolyester

ABSTRACT

Process for producing a copolyester of desired quality at low cost with high productivity, preferably a process for producing P(3HB-co-3HH) of 4 mol % or more 3HH content with a productivity as high as 40 g/L or more. In particular, a process for producing a copolyester of 3HB and 3HH comprising culturing a microorganism with the use of, as a carbon source, a fat or oil containing lauric acid as a constituent fatty acid under such conditions that phosphorus as a source of nutrition is limited.

This is a 371 national phase application of PCT/JP2003/013021 filled 10Oct. 2003, claiming priority to Japanese Application No. 2002-297602filed 10 Oct. 2002, the contents of which are incorporated herein byreference.

TECHNICAL FIELD

The present invention relates to a method for producing a copolyestercomprising 3-hydroxybutyric acid (hereinafter, referred to briefly as“3HB”) and 3-hydroxyhexanoic acid (hereinafter, referred to briefly as“3HH”) as monomeric units by using a microorganism.

BACKGROUND ART

Up to now, a large number of microorganisms have been known to storepolyester as an energy source substance within cells. A typical exampleof the polyester is poly-3-hydroxybutyric acid (hereinafter referred tobriefly as “P(3HB)”). P(3HB) is a thermoplastic polymer and isbiodegradable in the natural environment and, thus, has recentlyattracted attention as an ecofriendly green plastic. However, sinceP(3HB) is high in crystallinity, it is hard and fragile, so that therange of practical application thereof is limited. Therefore, studieshave been undertaken to modify the P(3HB) for improving theseproperties.

In the course of the study, a copolymer P(3HB-co-3HV) derived from3-hydroxybutyric acid (3HB) and 3-hydroxyvaleric acid (hereinafterreferred to briefly as “3HV”), and a production method thereof have beendeveloped (Japanese Kokai Publication Sho-57-150393, Japanese KokaiPublication Sho-59-220192 and Japanese Kohyo Publication Hei-11-500008).This P(3HB-co-3HV) is rich in flexibility as compared with P(3HB), henceit was considered to have a wide application range.

Methods for producing the copolymer P(3HB-co-3HV) described in thesepatent documents comprise growing cells in the first stage and culturinga microorganism with restricting nitrogen or phosphorus in the latterstage to produce the copolymer similarly to the conventional methods forproducing P(3HB).

Moreover, as for P(3HB-co-3HV), since the flexibility changes as acontent of 3HV increases, researches for controlling the 3HV contenthave also been made. For example, propionic acid is used in JapaneseKokai Publication Sho-57-150393 and Japanese Kokai PublicationSho-63-269989, and propan-1-ol is used in Japanese Kokoku PublicationHei-7-79705, and by changing an addition amount thereof to a medium, the3HV content in P(3HB-co-3HV) is controlled to produce P(3HB-co-3HV)having the 3HV content of 10 to 90 mol %.

Actually, however, P(3HB-co-3HV) shows only slight changes in thecharacteristics even when the 3HV content is increased. In particular,the flexibility is not improved to such an extent required for its usein films and the like. Thus, it has been used only in the field of rigidshaped articles such as shampoo bottles and disposable razor grips.

Under such circumstances, for making up the above-mentioned drawbacks ofthe copolymer derived from 3HB and 3HV, copolymers containing, as acomponent, a hydroxyalkanoic acid other than 3HB and 3HV such as3-hydroxypropionic acid (hereinafter referred to briefly as “3HP”),3-hydroxyhexanoic acid (hereinafter referred to briefly as “3HH”),3-hydroxyoctanoic acid (hereinafter referred to briefly as “3HO”),3-hydroxynonanoic acid (hereinafter referred to briefly as “3HN”),3-hydroxydecanoic acid (hereinafter referred to briefly as “3HD”) or3-hydroxydodecanoic acid (hereinafter referred to briefly as “3HDD”) areintensively studied (Poirier, Y., Nawrath C., Somerville C,BIO/TECHNOLOGY, 13, 142–150, 1995).

Among them, noteworthy studies are those on a copolyester comprising 3HBand 3HH units, particularly a copolymer P(3HB-co-3HH) derived only from3HB and 3HH, and on a production method thereof (Japanese KokaiPublication Hei-05-93049 and Japanese Kokai Publication Hei-07-265065).The production methods of copolyesters such as P(3HB-co-3HH) describedin these patent documents comprise a fermentation production from fattyacids such as oleic acid or oils and fats such as olive oil by usingAeromonas caviae isolated from soil.

A study regarding characteristics of P (3HB-co-3HH) has also beenconducted (Y. Doi, S. Kitamura, H. Abe, Macromolecules 28, 4822–4823,1995). This document reports a fermentation production of P (3HB-co-3HH)with a 3HH content of 11 to 19 mol % by culturing A. caviae with a fattyacid of not less than 12 carbon atoms. The result shows that, as the 3HHcontent increases, P(3HB-co-3HH) exhibits a gradual increase offlexibility from the hard and brittle characteristics of P (3HB) andfinally shows more flexibility than P(3HB-co-3HV).

Additionally, it was reported that a polyhydroxyalkanoic acid(PHA)synthase gene from A. caviae was cloned and introduced into R. eutrophahaving an accumulating ability of polyhydroxybutyric acid(PHB) of notless than 90% to generate a recombinant strain, which was then used toproduce P (3HB-co-3HH) using fatty acids as a carbon source (T. Fukui,Y. Doi, J. Bacteriol., vol. 179, No. 15, 4821–4830, 1997 and JapaneseKokai Publication Hei-10-108682). In these documents, it is reportedthat P (3HB-co-3HH) having the 3HH content of 10 to 20 mol % may beproduced by using sodium octanoate as a carbon source.

Furthermore, a method has been recently disclosed which comprises usingmultiple carbon sources in producing a polyester using the aboverecombinant strain, and it was revealed that a carbon number of an oilor fat or a fatty acid used as a carbon source had an influence on the3HH-content of P(3HB-co-3HH) (Japanese Kokai Publication 2001-340078).

If the 3HH content of P(3HB-co-3HH) can be controlled optionally in awide range in the future, both hard copolymers and soft copolymers maybe produced by fermentation, and such copolymers will find a broad rangeof applications, from chassis for TV-set, which is required to be hard,to a thread or a film, which are required to be flexible.

However, when a practical application of P(3HB-co-3HH) is considered,what becomes a barrier is the production cost. For example, in anymethods which have already been disclosed, the productivity ofP(3HB-co-3HH) is low and it is as much as 30 g/L. Additionally, a fattyacid having the carbon number of not less than 12, which is expensive asa carbon source, is used as the only carbon source, or an addition of anexpensive fatty acid (hexanoic acid) is required to improve the 3HHcontent. Thus, it is scarcely possible to apply such technologies to anindustrial production method of said polymer.

As described above, characteristics of P(3HB-co-3HH) are remarkablyaffected by the 3HH content. As a result of the investigation conductedby the present inventors, it is preferable to secure not less than 4 mol% of the 3HH content in order to enable wide applications ofP(3HB-co-3HH). However, in the conventional culture methods, not only anexpensive carbon source is required but also the productivity tends tobe more deteriorated when trying to improve the 3HH content (seeJapanese Kokai Publication 2001-340078).

Whereupon, it has been long awaited to develop a technology forrealizing a high productivity of cells and polymer content in low cost,and from an industrial point of view, a technology capable of producingP(3HB-co-3HH) having the higher productivity, and preferably, atechnology capable of producing P(3HB-co-3HH) having not less than 4 mol% of the 3HH content while securing that productivity.

SUMMARY OF THE INVENTION

In view of the above-mentioned state of the art, the present inventionprovides a method for producing P(3HB-co-3HH) which realizes a highproductivity in low cost, and preferably a method for producingP(3HB-co-3HH) having not less than 4 mol % of the 3HH content whilesecuring that productivity.

The present inventors have made various investigations, and particularlyhave studied on various fermentation materials (carbon sources) in viewof prices, supply stability, quality stability, yield of cells orpolymers and the like. As a result, they have succeeded in maintainingthe high productivity by culturing a microorganism accumulatingP(3HB-co-3HH) in a medium with an inexpensive oil or fat as a carbonsource, and further selecting the species of the oil or fat to be usedas a carbon source and culturing conditions. Moreover, they have alsosucceeded in achieving the 3HH content of not less than 4 mol %, whichis the desired value, by further restricting the content of specificoils and fats.

That is, the point of the present invention relates to a method forproducing a copolyester which secures the high productivity of not lessthan 40 g/L by restricting phosphorus, a nutrient source and using anoil or fat containing lauric acid in constituent fatty acids as a carbonsource in producing a copolyester derived from 3HB and 3HH, such asP(3HB-co-3HH), using a microorganism. Furthermore, the present inventionrelates to a method for producing a copolyester having the 3HH contentof preferably not less than 4 mol % by further restricting the contentof lauric acid in constituent fatty acids of the oil or fat to be usedas a carbon source to be not less than 10% by weight.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is a method for producing a copolyester comprisingat least 3-hydroxybutyric acid and 3-hydroxyhexanoic acid as monomericunits which comprises culturing an oil or fat containing lauric acid inconstituent fatty acids as a carbon source under a condition phosphorus,a nutrient source, being restricted.

The method for producing the copolyester according to the presentinvention is applied in producing a copolyester comprising 3HB and 3HHunits as monomeric units such as P(3HB-co-3HH) using a microorganism.

The copolyester produced by the method of the present invention is apolyester comprising at least 3HB and 3HH as a monomeric unit, and itmay also contain a monomeric unit other than 3HB and 3HH. As the thirdmonomeric unit in this case, there may be mentioned 3HV, 3HP, 3HO, 3HN,3HD and 3HDD, etc. However, in order to copolymerize monomeric unitshaving odd number of carbon chains such as 3HV, it is required to add acarbon source having odd number of carbon chains, which scarcely existsin natural oils and fats available as an inexpensive carbon source inthe culture method described below.

From an advantageous point of view such as a culture cost or othermerits on a process, it is preferable to use P(3HB-co-3HH) derived onlyfrom 3HB and 3HH as a copolyester produced by the method according tothe present invention.

When the production method of the present invention is used, theproductivity of the copolyester by a microorganism becomes not less than40 g/L. In the present specification, “the productivity of acopolyester” is represented by a weight of the produced copolyester in avolume of medium fluid (medium+microorganism+other by-product) at thetime of completion of the culture.

The content of 3-hydroxyhexanoic acid in the copolyester produced by theproduction method of the present invention is preferably not less than 4mol %.

It is further preferred that the productivity of the copolyester by amicroorganism is not less than 40 g/L and the content of3-hydroxyhexanoic acid in the copolyester is not less than 4 mol % inthe production method of the present invention.

In the production method according to the present invention, there is noparticular restriction on a microorganism to be used. For example, theremay be used microorganisms isolated from nature or those deposited todeposit authorities of strains (e.g. IFO, ATCC, etc.) and bacteria ofgenus Alcaligenes, genus Ralstonia, genus Aeromonas, genus Pseudomonas,genus Escherichia and the like. Among them, preferred is Ralstoniaeutropha.

Moreover, in the case that a wild-type of the above microorganismscannot produce an objective copolymer, or the case that the productionamount is low, the above microorganisms may be used in the form of atransformed microorganism after being transformed using a recombinantvector containing a polyester polymerase gene. As the vector used inproducing the transformed microorganism, there may be used a plasmidvector and the like which is able to grow within the cell autonomously.Also, said polyester polymerase gene may be directly incorporated intothe chromosome of the microorganism to be a host.

As a microorganism to be the host mentioned above, there may be usedbacteria such as one belonging to the genus Alcaligenes, genusRalstonia, genus Aeromonas, genus Pseudomonas or genus Escherichia.

The polyester polymerase gene to be used in the method for producing apolyester according to the present invention is not particularlyrestricted. However, preferred is a gene isolated from Aeromonas caviae,and for example, there may be used a gene fraction described in JapaneseKokai Publication Hei-10-108682 and the like.

The conventional methods may be applied in introducing the recombinantvector to the microorganism. For example, the conjugation method, thecalcium method, the electroporation method and the like may be used.

As a preferable example of the microorganisms to be used in the presentinvention, there may be mentioned a Ralstonia eutropha strainPHB-4/pJRDEE32d13, which was obtained by introducing a polyesterpolymeraze gene derived from Aeromonas caviae into Ralstonia eutropha(T. Fukui., Y. Doi., Appl. Microbiol. Biotechnol., 49, 333–336 (1998)).This strain is deposited to the National Institute of AdvancedIndustrial Science and Technology, International Patent OrganismDepositary (Central 6, 1-1-1, Higashi, Tsukuba, Ibaraki, JAPAN) on thedate of Aug. 7, 1997 under the name of Alcaligenes eutrophus AC 32 in anaccession number of FERM BP-6038 under Budapest treaty.

In the production of the copolyester of the present invention, aninexpensive oil or fat containing lauric acid is used as a carbon sourcefrom viewpoints of price, supply stability, quality stability, yield ofcells or polymers, and the like.

A medium containing a nitrogen source, inorganic salts, vitamins andother general organic nutrient sources may be used as a nutrient sourceother than the carbon source.

As the nitrogen source to be used in the production method according tothe present invention, there may be mentioned inorganic nitrogen sourcese.g. ammonia, ammonium salts such as ammonium chloride, ammonium sulfateand ammonium phosphate, and organic nitrogen sources such as peptone,meat extract and yeast extract, for instance.

As the inorganic salts, there may be used phosphates such as potassiumdihydrogenphosphate, dipotassium hydrogenphosphate or magnesiumphosphate; magnesium sulfate, sodium chloride and the like, forinstance.

As the vitamins, there may be used vitamin B1, vitamin B12, vitamin C,and the like.

As the other organic nutrient sources, there may be used amino acidssuch as glycine, alanine, serine, threonine and proline. However, from aviewpoint of controlling the production cost, it is preferable to useonly a minimum amount of organic nitrogen sources of peptone, meatextract and yeast extract, vitamins of vitamin B1, vitamin B12 andvitamin C, and organic nutrient sources of glycine, alanine, serine,threonine and proline. Particularly among them, the amount to be used ofpeptone, yeast extract and meat extract is more preferable to be kept ina minimum amount.

Not as long as P(3HB-co-3HH), it is generally said that, in producing apolyester by a microorganism, the polyester is preferably accumulatedwithin cells when a concentration of nutrients essential for growingsuch as nitrogen, phosphorus, and the like becomes low and an excessamount of carbon source exist in a medium. However, it has never beeninvestigated in detail for what kinds of nutrient restrictions arepreferable.

The present inventors have found that the productivity of P(3HB-co-3HH)becomes low in the case that nitrogen is restricted as compared with thecase that phosphorus is restricted, and bacteriolysis is caused underthe restriction of nitrogen although it depends on culture conditions.Therefore, in the method for producing the copolyester according to thepresent invention, phosphorus-restriction culture is adopted withoutrestricting nitrogen.

In the present invention, the method of restricting phosphorus duringthe culture is not particularly restricted, and the conventionalphosphorus-restriction culture conditions may be adopted. Moreover,needless to say, “restriction of phosphorus” in the present inventiondoes not mean a condition that completely no phosphorus atom iscontained in a medium but means a condition in which phosphorus as anutrient source is contained in a minimum amount necessary for growing.That is, it means a condition with a growing amount of cells beingdefined by phosphorus, and does not exclude phosphorus contained in asmall amount as an inorganic salt in a medium.

Generally, the oil or fat to be used for a fermentation by themicroorganism may include natural oils which are supplied in arelatively stable manner such as soybean oil, corn oil, cottonseed oil,palm oil, palm kernel oil, coconut oil, peanut oil and rapeseed oil,respective fractions obtained by fractioning these oils, for example,fractional oils called in an oils and fats industry by the names of“palm W olein oil” (low-melting point fraction obtained by fractionatingpalm oil twice without solvent), “palm kernel olein oil” (low-meltingpoint fraction obtained by fractionating palm kernel oil once withoutsolvent) or the like, and further a mixed oil obtained by mixing theseoils. However, in the production method of the present invention, it ispreferable to use an oil or fat containing lauric acid in constituentfatty acids, for example, an oil called as “lauric oils” in the oils andfats industry.

As the oil or fat containing lauric acid in constituent fatty acids mayinclude, for example, natural oils such as palm kernel oil and coconutoil, and respective fractions obtained by fractionating these oils, forexample fractional oils such as palm kernel olein oil, and it ispreferable to use a natural or fractionated lauric oils. Additionally,there is no problem to use an oil or fat in which lauric acid isintroduced in constituent fatty acids by treating an oil or fatcontaining no lauric acid chemically or biochemically, or the one inwhich the content of lauric acid is increased by treating a naturallauric oil biochemically. Furthermore, there may be used a mixed oilobtained by mixing 2 or more species of oils and fats containing lauricacid in constituent fatty acids, or a mixed oil obtained by mixing anoil or fat containing lauric acid in constituent fatty acids and an oilor fat containing no lauric acid in constituent fatty acids.

As the above carbon source, saccharides such as glucose, fructose,sucrose and lactose may be used together with an oil or fat containinglauric acid in constituent fatty acid in the production of thecopolyester according to the present invention.

As the content of lauric acid in constituent fatty acids of the oil andfat to be used in the present invention (in the case that a mixed oil isused, it is calculated as a total content in constituent fatty acids ofthe mixed oil) is preferably not less than 10% by weight, and morepreferably not less than 20% by weight. It is preferable to make thelauric acid content in constituent fatty acids of the oil and fat be notless than 10% by weight for securing the productivity of not less than40 g/L and for producing a copolyester having not less than 4 mol % ofthe 3HH content.

As an addition mode of the oil and fat, any modes such as a shot of alarge amount at once, addition in portions and continuous(stepwise) feedmay be applied in the present invention. However, as a result of theinvestigation conducted by the present inventors, it was found that whena large amount is shot at once, toxicity for cells may appear by the oilor fat, which is water-insoluble components in a medium, or by fattyacids generated by hydrolysis of the oil or fat by lipase, or an actualoperation may become difficult owing to foaming attributed to the fattyacids generated. Therefore, the oil or fat as a carbon source ispreferably added in portions little by little, or fed continuously orintermittently by using a pump and the like.

According to the current technologies, it is difficult to accuratelymeasure an amount of an oil or fat of water-insoluble components in amedium, or fatty acids generated by hydrolysis of an oil or fat bylipase either on-line or off-line. Thereupon, the present inventors haveobtained a feed pattern, in which supply of an oil or fat is notinsufficient but is not excess as causing a foaming, empirically frommany experiments using a jar fermentor and developed a method adoptingthis pattern as a basic guideline. In an actual operation, it ispreferable to apply a method comprising supplying the oil or fat withfeeding along this guideline, sampling at every certain period,centrifuging, and adjusting the feeding amount with observing thethickness of an oil or fat layer in the culture supernatant.

As a result of the investigation conducted by the present inventors, itwas confirmed that the guideline thus produced may also be appliedefficiently in a culture using a large fermentor.

As another indirect method, there maybe mentioned a method comprisingmeasuring an oxygen concentration and a carbon dioxide concentration inan exhaust gas to obtain an oxygen consuming rate or a carbon dioxidegeneration rate. Using these values as an index, a feed rate of the oilor fat may be changed. In this case, it is preferable to study arespiration property of cell growing period and polyester productionperiod in detail beforehand and make an adjustment.

In the production method according to the present invention, the rangeof the 3HH content of the produced copolyester may be controlledoptionally due to a species of the oil or fat to be added as a carbonsource. For example, in the cases that the lauric oils such as coconutoil, palm kernel oil or a fractional oil or fat of said oils and fatsare used alone or in admixture as the carbon source, P(3HB-co-3HH)having relatively high 3HH content of 4 to 20 mol % may be obtained. Inthe cases that mixed oils containing soybean oil, corn oil, cottonseedoil, palm oil, peanut oil, rapeseed oil or a fractional oil of theseoils, which contain no lauric acid in constituent fatty acids, and theabove lauric oils are used, P(3HB-co-3HH) having relatively low 3HHcontent of not more than 10 mol % may be produced.

Moreover, by adjusting the mixing ratio of said mixed oils to change thelauric acid content in constituent fatty acids, multiple species of P(3HB-co-3HH) having an optional 3HH content may be produced whilemaintaining the high productivity of not less than 40 g/L. Furthermore,to produce a copolymer having a third component other than 3HB and 3HH,a carbon source corresponding to the third component, for example afatty acid having odd number of carbon chains may be added to the oiland fat containing lauric acid in the above constituent fatty acids.

The culture temperature of the microorganism may be any temperatures aslong as the cells may grow, but preferably at 20 to 40° C., morepreferably at 25 to 35° C. The culture period is not particularlyrestricted and may be 1 to 7 days, preferably 40 to 70 hours.

The above explained restriction of the species of the oil or fat,restriction of phosphorus or the like in producing the polyester iscarried out in a main culture in a polyester production medium.Incidentally, the microorganisms are generally cultured in a seed mediumor a preculture medium to grow cells to a certain level prior to themain culture with the polyester production medium. In such a case, thesame nutrient sources as described above maybe used in the seed mediumor the preculture medium. The culture temperature in these media may bethe same as the above polyester production medium, and the cultureperiod is preferably 1 to 2 days, respectively.

Additionally, when the transformed microorganism is used as amicroorganism, for example, antibiotics such as kanamycin, ampicillinand tetracycline, which correspond to a resistant gene exist in vectorsand the like, may be added to a preculture medium.

In the production method of the present invention, a method forcollecting the copolyester from the microorganism is not particularlyrestricted, and the conventional solvent extraction methods, physicalcell disruption, and chemical treatment, etc. may be used and thefollowing methods may also be used, for instance. After completion of aculture, cells are separated from a culture broth by a centrifuge andthe like, then the cells are washed with distilled water, methanol andthe like, and dried. A polyester is extracted from the dried cells usingorganic solvents such as chloroform. Cell components are removed fromthe polyester-containing organic solvent by filtration, etc., and poorsolvents such as methanol and hexane are added to the filtrate andprecipitate the polyester. The supernatant is removed by filtration orcentrifugation, and dried to collect a polyester.

The monomeric unit of the polyester obtained can be analyzed by gaschromatography or nuclear magnetic resonance spectrometry, for instance.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a guideline showing a feed pattern in Example 1, 4, 5 andComparative Example 1.

BEST MODE FOR CARRYING OUT THE INVENTION

The following examples illustrate the present invention morespecifically. These examples are, however, by no means limitative of thetechnical scope of the present invention.

EXAMPLE 1 AND COMPARATIVE EXAMPLE 1

Ralstonia eutropha strain PHB-4/pJRDEE32d13 (T. Fukui., Y. Doi., Appl.Microbiol. Biotechnol., 49, 333–336 (1998)) (hereinafter, referred tobriefly as “Red 13” strain) was cultured as follows. Incidentally, asdescribed above, said Red 13 strain is deposited to the NationalInstitute of Advanced Industrial Science and Technology, InternationalPatent Organism Depositary (Central 6, 1-1-1, Higashi, Tsukuba, Ibaraki,JAPAN)on the date of Aug. 7, 1997 under the name of Alcaligeneseutrophus AC 32 in an accession number of FERM BP-6038 under Budapesttreaty.

The composition of the seed medium was made to comprise 1 w/v %Meat-extract, 1 w/v % Bacto-Trypton, 0.2 w/v % Yeast-extract, 0.9 w/v %Na₂PO₄□12H₂0, 0.15 w/v % KH₂PO₄ and pH 6.8.

The composition of the preculture medium was made to comprise 1.1 w/v %Na₂PO₄□12H₂O, 0.19 w/v % KH₂PO₄, 1.29 w/v % (NH₄)₂SO₄, 0.1 w/v %MgSO₄□7H₂O, 2.5 w/v % palm W olein oil, 0.5 v/v % trace metal saltsolution (1.6w/v % FeCl₃□6H₂O, 1 w/v % CaCl₂□2H₂O, 0.02 w/v %CoCl₂□6H₂O, 0.016 w/v % CUSO₄□5H₂O and 0.012 w/v % NiCl₂□6H₂O weredissolved in 0.1 N hydrochloric acid), and 5×10⁻⁶ w/v % kanamycin.

The composition of the polyester production medium was made to comprise0.385 w/v % Na₂PO₄□12H₂O, 0.067 w/v % KH₂PO₄, 0.291 w/v % (NH₄)₂SO₄, 0.1w/v % MgSO₄□7H₂O, 0.5 v/v % trace metal salt solution (1.6 w/v %FeCl₃□6H₂O, 1 w/v % CaCl₂□2H₂O, 0.02 w/v % CoCl₂□6H₂O, 0.016 w/v %CuSO₄□5H₂O and 0.012 w/v % NiCl₂□6H₂O were dissolved in 0.1 Nhydrochloric acid) and 0.05 w/v % BIOSPUREX 200K (antifoaming agent:product of Cognis Japan Ltd.)

A glycerol stock (50 μl) of Red 13 strain was inoculated to a seedmedium (10 ml) and cultured for 24 hours. Then, the resultant wasinoculated in a ratio of 0.2 v/v % to a 5 L jar fermentor (MDL-500 type,product of B.E. Marubishi Co., Ltd.) containing 3 L of the preculturemedium. The running condition was set to be a culturing temperature of30° C., a stirring rate of 500 rpm, an aeration rate of 1.8 L/min. Theculture was carried out for 28 hours with controlling pH between 6.7 and6.8 to obtain a culture seed. 7% ammonium hydroxide solution was usedfor the pH control.

The polyester production culture was carried out as the following. Nineof 10 L jar fermentors (MDL-1000 type, product of B.E. Marubishi Co.,LTD) containing 6 L of the production medium were made ready, and 1.0v/v % of the preculture seed was inoculated to the respective media.Then, 9 species of oils and fats, that is, soybean oil, cottonseed oil,rapeseed oil, corn oil, palm W olein oil, peanut oil, coconut oil, palmkernel oil and palm kernel olein oil were added to the respective jarfermentors. These oils and fats were added to an initial medium at aratio of 1 w/v %, and fed as following the guideline shown in FIG. 1 (afeed pattern in which supply of the oil or fat is not insufficient butis not excess). Incidentally, the unit “(ml/hour)/L” of feed rate of theoil or fat in FIG. 1 represents a feed rate (ml) of the oil or fat per 1L of the culture broth by hour. The running condition was set to be aculturing temperature of 28° C., a stirring rate of 400 rpm, an aerationrate of 3.6 L/min and pH was controlled of between 6.7 and 6.8. Theculture was carried out for 60 hours, and after completion of theculture, cells were collected by a centrifugation, washed with methanoland lyophilized to measure the weight of the dried cells.

To approximately 1 g of the dried cell obtained was added with 100 ml ofchloroform, and the mixture was stirred overnight at a room temperatureto extract a polyester within cells. After the cell residue was filteredoff, the resultant was concentrated by an evaporator until the totalcontent to be 30 ml, approximately 90 ml of hexane was added graduallywith stirring slowly, and allowed to stand for 1 hour. The precipitatedpolyester was filtered off, and dried in vacuo for 3 hours at 50° C. Theweight of the dried polyester was measured and the content of thepolyester within the cells were calculated.

Approximately 20 mg of the obtained dried polyester was added with 2 mlof a sulfuric acid-methanol mixture (15:85) and 2 ml of chloroform, andthe container was sealed. The obtained mixture was heated at 100° c. for140 minutes to obtain methylester of a polyester decomposition product.After cooling, 1.5 g of sodium bicarbonate was added by little andlittle to neutralize, and the mixture was left until generation ofcarbon dioxide stops. After 4 ml of diisopropyl ether was added andwell-mixed, the mixture was centrifuged to analyze a composition ofhydroxyalkanoic acid methyl ester of the polyester decomposition productin a supernatant by a capillary gas chromatography, and a composition(content) of a monomer unit of the obtained polyester was determined.The used gas chromatograph was GC-17A produced by Shimadzu Corporation,and the used capillary column was NEUTRA BOND-1 produced by GL ScienceCo., LTD (column length 25 m, column inner diameter 0.25 mm and liquidfilm thickness 0.4 μm). The temperature raising rate was set to be 8°c./min from 100° c. of an initial temperature to 200° c., and further30° c./min from 200° c. to 290° c.

The effects given by differences of lauric acid content in respectiveoils and fats and species of the oils and fats used as carbon sources ona 3HH mole fraction ratio of P(3HB-co-3HH) and the productivity wereshown in Table 1. Table 1 shows the results of the culture at 60-hour.

TABLE 1 Lauric acid con- Productivity tent in the of P(3HB- 3HH used oilco-3HH) content (weight Oils and fats (g/L) (mol %) %) ComparativeSoybean oil 62 3.0 0 Example 1 Cottonseed oil 56 2.5 0 Rapeseed oil 522.7 0 Corn oil 68 2.7 0 Palm W olein oil 62 3.0 0 Peanut oil 45 3.6 0Example 1 Coconut oil 42 13.8   47 Palm kernel oil 48 6.8  47 Palmkernel olein oil 71 7.9  41

From the results, it was found that when coconut oil, palm kernel oil orpalm kernel olein oil, which contain lauric acid in constituent fattyacids, were used, the 3HH contents of the polyester obtained were ashigh as about 7 to 14 mol %, and the 3HH contents of all the other fatscontaining no lauric acid in constituent fatty acids were lower than 4mol %. It was found that the 3HH contents, which has a remarkable effecton qualities and characteristics of the polyester, differ significantlydepending on the difference of the oil or fat used as a substrate andpresence or absence of lauric acid. Although the lauric oils other thanpalm kernel olein oil were slightly low in the productivity of thepolyester compared with the other oils and fats, the productivitycapable of being applied for an industrial production, that is not lessthan 40 g/L, was secured.

EXAMPLE 2 AND COMPARATIVE EXAMPLE 2

Using the same oil as of Example 1 and Comparative Example 1, the strainwas cultured in the same manner as in Example 1 and Comparative Example1 except that the respective oils and fats are added in a ratio of 3 w/v(%) into an initial medium and in a ratio of 2 w/v (%) at every 12 hoursup to 48 hours. The results of the culture at 60-hour are shown in Table2.

TABLE 2 Productivity 3HH of P(3HB-co-3HH) content Oils and fats (g/L)(mol %) Comparative Soybean oil 64 2.5 Example 2 Cottonseed oil 60 2.3Rapeseed oil 56 2.1 Corn oil 71 2.2 Palm W olein oil 65 2.8 Peanut oil45 3.8 Example Coconut oil 45 10.4  2 Palm kernel oil 48 5.9 Palm kernelolein oil 72 5.8

Foams were generated more vigorously than Example 1 and ComparativeExample 1 throughout the cultures in any oils and fats. However, thehigh productivity of not less than 40 g/L and the desired 3HH content ofnot less than 4 mol % were obtained when coconut oil, palm kernel oil orpalm kernel olein oil was used, which contains a large amount of lauricacid, also in the cases where the oils and fats were added in portions.

EXAMPLE 3 AND COMPARATIVE EXAMPLE 3

Using the same oil as of Example 1 and Comparative Example 1, the strainwas cultured in the same manner as in Example 1 and Comparative Example1 except that the respective oils and fats were fed at a constant feedrate of 15 ml/h from 0 to 57-hour. The results of the culture at 60-hourare compared in Table 3.

TABLE 3 Productivity 3HH of P(3HB-co-3HH) content Oils and fats (g/L)(mol %) Comparative Soybean oil 60 2.7 Example Cottonseed oil 54 2.7 3Rapeseed oil 50 2.5 Corn oil 66 2.3 Palm W olein oil 64 2.7 Peanut oil42 3.9 Example Coconut oil 43 9.6 3 Palm kernel oil 45 5.6 Palm kernelolein oil 70 6.0

Foams were generated more vigorously than Example 1 and ComparativeExample 1 in the middle stage of the culture in any oils and fats.However, the high productivity of not less than 40 g/L was obtained whencoconut oil, palm kernel oil and palm kernel olein oil were used, whichcontain large amount of lauric acid, also in the cases where the oilsand fats were fed at a constant rate while securing the 3HH content ofnot less than 4 mol %.

EXAMPLE 4

The strain was cultured in the same medium and condition as in Example 1and Comparative Example 1 except that mixed oils were used, whichcomprise palm kernel olein oil and coconut oil in 1:1 (v/v), palm kernelolein oil and peanut oil in 1:1 (v/v), palm kernel olein oil and soybeanoil in 1:1 (v/v) and palm kernel olein oil and corn oil in 1:1 (v/v),and the results shown in Table 4 were obtained. The results of theculture at 60-hour are compared in Table 4.

TABLE 4 Productivity of 3HH P(3HB-co-3HH) content Mixed oils (g/L) (mol%) Palm kernel olein oil 1:1 (v/v) 68 8.5 and coconut oil Palm kernelolein oil 1:1 (v/v) 65 6.2 and peanut oil Palm kernel olein oil 1:1(v/v) 72 5.2 and soybean oil Palm kernel olein oil 1:1 (v/v) 67 4.5 andcorn oil

As a result of increasing the lauric acid content in substrate oils andfats by using the mixed oils containing a lauric oil, P(3HB-co-3HH)having the 3HH content of not less than 4 mol % were produced even whencorn oil and soybean oil were used, which produced P (3HB-co-3HH) havingthe 3HH content of not more than 3 mol % in Comparative Example 1.

In the case of peanut oil and coconut oil, by using the oils in theforms of mixed oils, the productivities were improved, and the 3HHcontent showed a medium value of the respective oils and fats beforemixing.

EXAMPLE 5

The strain was cultured in the same medium and condition as in Example 1and Comparative Example 1 except that 4 species of mixed oils in whichthe mixing ratio of palm kernel olein oil and soybean oil was changedwere used as a substrate, that is, mixed oil A (palm kernel oleinoil/soybean oil=75/25 (v/v)), mixed oil B (palm kernel olein oil/soybeanoil=50/50(v/v)), mixed oil C (palm kernel olein oil/soybean oil=25/75(v/v)) and mixed oil D (palm kernel olein oil/soybean oil=20/80 (v/v)).The results shown in Table 5 were obtained. The results of the cultureat 60-hour are compared in Table 5.

TABLE 5 Lauric acid con- Productivity tent in the of P(3HB- 3HH used oilco-3HH) content (weight Mixed Oils (g/L) (mol %) %) Mixed oil A (Palmkernel olein oil/ 68 6.1 30.8 Soy bean oil = 75/25 (v/v)) Mixed oil B(Palm kernel olein oil/ 72 5.2 20.5 Soybean oil = 50/50 (v/v)) Mixed oilC (Palm kernel olein oil/ 70 4.7 10.3 Soybean oil = 25/75 (v/v)) Mixedoil D (Palm kernel olein oil/ 72 3.2  8.2 Soybean oil = 20/80 (v/v))

As shown in Table 5, differences were not significantly acknowledged inthe productivity depending on differences of the mixing ratio. However,the 3HH content became high as the ratio of palm kernel olein oil in themixed oils was large. Particularly, the mixed oils containing not lessthan 10% by weight of lauric acid could produce P(3HB-co-3HH) having thedesired 3HH content of not less than 4 mol % and expressing preferablecharacteristics.

INDUSTRIAL APPLICABILITY

In the method according to the present invention, P(3HB-co-3HH) havingthe desired 3HH content and expressing preferable characteristics may beproduced while securing the high productivity of not less than 40 g/L byrestricting phosphorus and using oils or fats containing lauric acid asa carbon source. Moreover, by making the content of lauric acid inconstituent fatty acids in the oils or fats to be not less than 10% byweight, P(3HB-co-3HH) having the 3HH content of not less than 4 mol %may be produced while securing the high productivity of not less than 40g/L. Accordingly, it becomes possible to produce or provideP(3HB-co-3HH) having a broad application range in the industrial field.

1. A method for producing a copolyester comprising at least3-hydroxybutyric acid and hydroxyhexanoic acid as monomeric units by amicroorganism which comprises culturing the microorganism with an oil orfat containing lauric acid in constituent fatty acids as a carbon sourceunder condition phosphorus, a nutrient source, being restricted, whereinthe lauric acid content in constituent fatty acids of the oil or fat is10 to 41% by weight.
 2. The method according to claim 1, wherein the oilor fat containing lauric acid is palm kernel olein oil.
 3. The methodaccording to claim 1, wherein the oil and fat used as a carbon source isa mixed oil obtained by mixing palm kernel olein oil and an oil or fatcontaining no lauric acid in constituent fatty acids.
 4. The methodaccording to claim 3, wherein the oil or fat containing lauric acid is amixed oil obtained by mixing palm kernel olein oil and at least one oilor fat selected from the group consisting of peanut oil, soybean oil andcorn oil.
 5. The method according to claim 1, wherein the microorganismis a transformed microorganism incorporated with a polyester polymerasegene isolated from Aeromonas caviae.
 6. The method according to claim 1,wherein the microorganism is Ralstonia eutropha.